Loss of endothelial barrier integrity leads to increased vascular permeability and alveolar flooding, and contributes to morbidity and mortality associated with ARDS. In endothelial cells, thrombin activates G protein-coupled receptors and induces actin stress fibers followed by intercellular gap formation, and resultant increased trans-endothelial permeability to protein and liquid. The activation of the alpha subunit of heterotrimeric G13 protein by thrombin in endothelial cells is critical for the activation of Rho proteins and assembly of actin stress fibers and the subsequent cell retraction and increased endothelial permeability responses. Our Supporting Data suggests that G-alpha13 protein interacts with the actin-binding protein, radixin, implicated in the assembly of focal adhesions and actin filaments. Radixin may be a critical effector in signaling the G-alpha 13-induced Rho activation via the Rho guanine nucleotide dissociation inhibitor, RhoGDI. We will address a new and potentially important signaling pathway for the G-alpha13-dependent Rho activation and the resultant increased lung endothelial permeability. We will define the upstream regulation of Rho involving radixin and the signaling pathways mediating the increase in endothelial permeability. Under physiological conditions, the thrombin-induced cytoskeleton alterations and increased endothelial permeability are short-lived (approximately 2 hr) and reversible. The reversibility of thrombin-induced action is critical in restoring endothelial barrier integrity; however, its molecular and cellular bases are poorly understood. Protein kinase A, PKA, a kinase linked to enhancing integrity of the endothelial barrier is usually activated by cyclic AMP. We have discovered that both thrombin and G-alpha 13 can stimulate PKA via two novel mechanisms that do not require cAMP. One mechanism is dependent on the interaction of G-alpha 13 with radixin. Another mechanism is dependent on stimulation of the NF-kappaB signaling pathway via mitogen-activated protein kinases. The PKA-dependent phosphorylation of G-alpha13 may thereby inhibit Rho activation, thus providing a mechanism for down regulation of G-alpha13 activity and reversal of the permeability response. Also, G-alpha 13 may induce phosphorylation of vasodilator-stimulated phosphoprotein, VASP, which may prevent actin polymerization and thus promote the re-establishment of endothelial junctional barrier. Thus, we will test the hypothesis that signaling complexes formed by PAR-1 activation of G-alpha 13 create the molecular basis for ligand-dependent loss of endothelial barrier function as well as its restoration. This proposal will address the molecular and cellular mechanisms that enable G-alpha 13 to form specific signaling complexes, thereby generating signals that both create either loss and the subsequent restoration of endothelial barrier function. We believe that the proposed studies will generate important and novel information elucidating lung endothelial barrier regulatory mechanisms and will identify novel targets for therapies whereby the inappropriate increase in endothelial permeability can be controlled to reduce the vascular "leak" associated with ARDS.